The route of administration of a particular vaccine is dependent upon several factors. Factors to be considered include locus of initiation of infection, locus of disease progression, systemic or nonsystemic involvement, pathogenicity of the particular vaccine used, and type of immunity desired to be induced. Vaccines have been given orally, parenterally, and through inhalation. Oral and inhalation administration of vaccines is preferable when it is desirous to stimulate production of secretory immunoglobulin as a first line of defense against infection. Oral administration of vaccines may also be preferable in the instance of a particularly pathogenic organism, even though the organism has been attenuated prior to administration.
As the pathogenicity of the particular organism administered as a vaccine increases, so do concerns regarding incomplete attenuation. Alternatives have been developed which utilize protein subunits of antigenic molecules expressed on the surface of the organisms instead of the complete organism. However, although such materials are generally antigenic, they are not always immunogenic. Immunogenicity depends upon, among other factors, size. Several attempts have been made to address the antigen immunogen dichotomy in particularly pathogenic organisms. In that regard, various adjuvants were developed to augment the immunogenicity of a particular antigen and include such materials as keyhole limpet hemocyanin, aluminum hydroxide gels, sodium alginate, synthetic polynucleotides, muramyl dipeptide, Bordetella pertussis, Freund's Complete Adjuvant (emulsion of mineral oil, water, and mycobacterial extracts, and Freund's Incomplete Adjuvant (emulsion of water and oil only). Liposomes have also been used as adjuvants.
A liposome is a lipid-containing vesicle capable of entrapping various molecules of interest. Previously, most liposome vesicles functioned as adjuvants on the principle of entrapment of the antigen within a central core. However, liposomes have been developed which attempt to integrate the antigen within the lipid bi-layer.
U.S. Pat. No. 5,100,662 issued to Bolcsak et al. on Mar. 31, 1992 describes liposomes or liposome-like structures comprising sterols either alone or in combination with additional liposome-forming lipids. Liposome structures such as micelles, reverse micelles, hexagonal phases, multilamellar vesicles, or unilamellar vesicles are described. The liposomes may be prepared with or without the use of an organic solvent and may function as vaccines after entrapment or association of an immunogen.
While not specifically addressed to vaccine preparations, U.S. Pat. No. 5,049,388 issued to Gilbert et al. on Sep. 17, 1991 describes small particle aerosol liposomes and liposome-drug combinations for medical use and discloses that the drug or medication is interactive with the liposome membrane so that, on its rupture, the drug or medication is not lost from the liposome. Before aerosolization, the liposomes are heterogenous in size.
U.S. Pat. No. 4,900,549 issued to de Vries on Feb. 13, 1990, relates to a process for preparing immunogenic complexes. The patent describes an amphipathic antigenic protein or peptide contacted with a solution containing a detergent, a sterol, and a glycoside comprising hydrophobic and hydrophilic regions. Subsequently, the detergent is removed and the immunogenic complex is purified. Optionally, the solution further comprises a phospholipid, preferably phosphatidylethanolamine. The structure is described as consisting of cage-like or two-dimensional aggregates, depending upon whether phospholipid is or is not present, respectively.
The incorporation of pathogen subunits into liposome preparations for stimulation of immune response has been described previously. More specifically, the incorporation of a soluble antigen extract of Brucella abortus and the lipopolysaccharide (LPS) of Salmonella abortus equi, Escherichia coli, and Serratia marcescens. Fountain, et al., "Effect of Phosphatidylcholine Liposomes Containing Brucella abortus Soluble Antigen on the Response of Bovine Lymphocytes to Phytohemagglutinin," Current Microbiology, Vol. 6, pp. 61-64, 1981. These preparations involved incorporation of the antigens into a phosphatidylcholine liposome preparation described in Schuster et al., "Production of Antibodies Against Phosphocholine, phosphatidylcholine, Sphingomyelin and Lipid A by Injection of Liposomes Containing Lipid A," American Journal of Immunity, Vol. 122, pp. 4003-4009, 1979, and involved the entrapment of the subunit within the liposome.
Conventional liposome preparations, i.e., those entrapping the molecule of interest, are not satisfactory for vaccine formation for several reasons. They are generally heterogeneous in size and difficult to sterilize for in vivo applications. Their stability or shelf life is often very limited. Further, the selection of passenger molecules for entrapment may be limited.
A new liposome was developed consisting of an amphipathic vehicle called a solvent dilution microcarrier (SDMC) which enables the integration of the particular molecule into the bi-layer of the lipid vesicle itself, or in association with the component of the bi-layer, rather than inside the space created by the spherical bi-layer upon formation of the vesicle. The preparation of SDMCs is described in U.S. Pat. No. 5,133,965 issued to Michael W. Fountain on Jul. 28, 1992.
Specifically, U.S. Pat. No. 5,133,965 describes a method for preparing SDMCs exhibiting substantial size homogeneity and capable of being sterilized by a variety of methods, including heating or UV radiation.
It has been found that, through a modification of the method described in U.S. Pat. No. 5,133,965, the preparation of immunogenic complexes between SDMCs and normally non-immunogenic or weakly immunogenic antigens can be effected. Additionally, it has been found that immunogenic complexes between SDMCs and the subunits of pathogens can be formed. Further, the use of SDMCs facilitates the administration of the immunogens so prepared by a variety of means, including oral ingestion and nasal inhalation.
It has further been found that a shelf-stable SDMC precursor solution can be prepared and stored at room temperature until it is desired to add the antigenic material to be utilized in the vaccine. The SDMC precursor solution can be used in a method for remote encapsulation of other active ingredients as well, which active ingredients are inherently heat-labile, solvent intolerant or unstable over time. Thus, the SDMC precursor can be kept as a stock item and used to freshly prepare encapsulated actives of interest.